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Team:Brown/Notebook/July7
From 2010.igem.org
Wednesday, July 7 2010
Results of overnight plating:
- Empty pNoTat showed <5 colonies (suspect)
- Religated pGEM, pNoTat showed no growth.
- Control RFP showed colonies
Harvested 4 colonies from empty pNoTat plate.
Added to 5 mL of LB in culture tube, added 10 µl of Amp (100 mg/mL) to achieve total 200 µl/mL
pWill1 Glycerol Stocks
Added 750 µl culture, 250 µl glycerol per tube.
2 overnight tubes (1 and 2) stocked into 2 cyro tubes each.
LB Agar plates
- Mixed 12.5 g LB lennox broth with 7.5 g Agar and 500 mL dH2O
- Mixed 6.25 g LB lennox broth with 3.25 g Agar in 250 mL dH2O
- Cooked both until clear (not sure about concentrations)
Redo Ligation of WillRS and pGEM
- 7.5 µl 2X rapid ligation buffer
- 1.0 µl T4 DNA ligase
- 0.5 µl pGEM T Easy Vector
- 1.5 µl Will1 RS insert
- 4.5 µl dH2O
Made competent CaCl2 cells (Quick protocol)
Transferred Xl1 cells with ligation
Plated on amp+ plates, incubate at 37°C starting 7:00 PM
